The islet research of our laboratory has four interdependent objectives: 1) To continue exploring the glucokinase-glucose-sensor concept. This includes the study of kinetic and structural features of recombinant human wildtype glucokinase (GK) isoforms and selected mutants of MODY patients and further explorations of the physiological regulation of B-cell glucokinase activity emphasizing acute activation and slow induction by fuels and the role of feed back regulation by the GK regulator protein. 2) To continue exploring metabolic coupling in A- and B-cells. The roles of ATP, free ADP, of the ATP/ADP-free ratio and of the creatine kinase/P- creatine system will be investigated including the use of transgenic mice over-expressing creatine kinase in A- and B-cells. Transgenic mice expressing PEPCK in B-cells will be employed to explore the role of glycolytic intermediates in metabolic coupling with hexoses and pyruvate as stimuli. 3) To continue exploring the biochemical basis of B-cell adaptation in an organ culture system during chronic overload with various fuels including glucose, physiological mixtures of natural amino acids, fatty acids, ketone bodies and model fuels. A variety of techniques will be applied including the study of oxygen consumption, fuel metabolism, ion fluxes and the use of high resolution 2-D PAGE to identify and characterize fuel response proteins. 4) To begin addressing the issue of cellular heterogeneity of islet tissue with a state of the art computerized microscopic image analysis system allowing the investigation of fuel metabolism, Ca2+ levels and cAMP levels in identified and subtyped A- and B-cells during adaptation to fuels. Previous work from our laboratory has helped to set the stage for the present plans and we have the manpower, skills, collaborative backing and facilities to address these fundamentally important issue of islet research.